University of Louisville ThinkIR: The University of Louisville's Institutional Repository College of Arts & Sciences Senior Honors Theses College of Arts & Sciences 5-2018 Antibiotic resistant bacteria in water environments in Louisville, Kentucky. Amy Priest University of Louisville Follow this and additional works at: https://ir.library.louisville.edu/honors Part of the Environmental Microbiology and Microbial Ecology Commons Recommended Citation Priest, Amy, "Antibiotic resistant bacteria in water environments in Louisville, Kentucky." (2018). College of Arts & Sciences Senior Honors Theses. Paper 173. Retrieved from https://ir.library.louisville.edu/honors/173 This Senior Honors Thesis is brought to you for free and open access by the College of Arts & Sciences at ThinkIR: The University of Louisville's Institutional Repository. 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Antibiotic Resistant Bacteria in Water Environments in Louisville, Kentucky: An Analysis of Common Genera and Community Diversity By Amy Priest Submitted in partial fulfillment of the requirements for Graduation summa cum laude and for Graduation with Honors from the Department of Biology University of Louisville May, 2018 1 Table of Contents Abstract ........................................................................................................................................... 5 Introduction ..................................................................................................................................... 6 Antibiotic Resistance .................................................................................................................. 6 Aquatic Environments and Bacterial Diversity .......................................................................... 7 Human-Mediated Spread of Resistance in Natural Environments ............................................. 8 Vancomycin and Colistin ............................................................................................................ 9 Impoverished Areas and Antibiotic Resistance ........................................................................ 10 Project Rationale ........................................................................................................................... 11 Materials and Methods .................................................................................................................. 12 Collecting Antibiotic Resistant Isolates .................................................................................... 12 General and Selective Solid Culture Medium ...................................................................... 12 Sample Collection ................................................................................................................. 13 Preparing Antibiotic Resistant Samples for 16S rRNA Sequencing ........................................ 14 Genomic DNA Extraction .................................................................................................... 14 Amplifying 16S rRNA using Polymerase Chain Reaction ................................................... 14 Identifying Antibiotic Resistant Isolates ................................................................................... 15 Individual Isolate Sequencing and Database Analysis ......................................................... 15 Whole Community Analysis ..................................................................................................... 16 Metagenome Sequencing ...................................................................................................... 16 2 Group Comparisons .............................................................................................................. 17 Analysis of Community Locations and Income Data ........................................................... 17 Ochrobactrum sp. Analysis ...................................................................................................... 18 Phenotypic Differentiation of Isolates .................................................................................. 18 Genomic DNA Extraction and 16s rRNA Amplification ..................................................... 19 Minimum Inhibitory Concentration (MIC) Assays .................................................................. 20 Antibiotic Preparations ......................................................................................................... 20 Preparation of 96-well Plates ................................................................................................ 20 Assessing Ochrobactrum’s Ability to Transfer Resistance ...................................................... 22 Plasmid Determination ......................................................................................................... 22 Mating Assays ....................................................................................................................... 22 Results ........................................................................................................................................... 23 Identifying Antibiotic Resistant Isolates ................................................................................... 24 Bacterial Isolates Taxonomic Designations .............................................................................. 29 Whole Community Analysis ..................................................................................................... 32 Obtaining Communities ........................................................................................................ 32 Diversity within the Community Samples at the Genus Level ............................................. 33 Selective and Non-Selective Community Genera Comparisons .......................................... 34 Community Species Classifications……………………………………………………………...37 Selective and Non-Selective Community Species Comparisons .......................................... 37 3 Group Comparisons: Alpha Diversity……………………………………………………...........39 Group Comparisons: Beta Diversity……………………………………………………………..42 Income and Diversity Comparisons .............................................................................................. 44 Ochrobactrum sp. Analysis………………………………………………………………………46 Phenotypic and 16S Sequencing Determination of Ochrobactrum Isolates ......................... 47 Understanding Ochrobactrum/Pseudochrobactrum antibiotic resistance profiles ............... 48 Transmission of antibiotic resistance by Ochrobactrum .......................................................... 55 Discussion ..................................................................................................................................... 56 Acknowledgements ....................................................................................................................... 61 References ..................................................................................................................................... 62 Appendix…………………………………………………………………………………………68 Supplementary Protocol 1…………………………………………………………..........69 Supplementary Figure 1………………………………………………………………….75 4 Abstract Antibiotic resistant bacterial strains are an increasing problem, particularly in clinical health care settings. As a result, bacterial infections are becoming increasingly challenging to treat with more cases becoming life threatening. Aquatic environments facilitate microbial diversity and the transfer of genetic elements and thus may serve as a reservoir for antibiotic resistant microbes. Human misuse of antibiotics may further facilitate the spread of resistance in water environments. With little known about the bacteria communities in local water environments, this study aimed to learn more about these populations through the following aims: 1) identify the microbial community composition from water environments around Louisville, KY; and 2) examine of the communities were resistant to two clinically used antibiotics—vancomycin and colistin. In this study, water sites were sampled and sorted into 4 categories: agricultural waters, commercial drains, natural waters, and wastewaters. In total, 155 single colony isolates resistant to vancomycin and colistin were identified through 16S sequencing. Whole community metagenomics analysis characterized the bacterial composition of 87 communities from the initial sample collection. Community diversity and the relationship between diversity and income was analyzed. One of the most striking results was the presence of Ochrobactrum sp. in 78 of the 87 communities. Two of the most prevalent genera, Ochrobactrum and Pseudochrobactrum, were characterized by assessing relative antibiotic resistance profiles and were found to be tolerant to high doses of a spectrum of antibiotics. Finally, a representative Ochrobactrum sp. isolate was tested for its ability to confer antibiotic resistance to a susceptible recipient bacterium. This Ochrobactrum sp. isolate was unable to transfer colistin resistance to another bacterial species, Pseudomonas aeruginosa, despite repeated efforts. The results indicate that there is a large diversity of microbes